This project is part of NOT-OD-09-058, with Notice Title, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications" and is consistent with the aims of parent grant DE06988 which seeks to determine if amelogenin proteins contribute to cell behavior by promoting signals that change differentiation. In this project we expand parent grant specific aim 3, to expand the scope of investigation into the role of amelogenin to include their capacity to induce an osteogenic pathway in stem cells. The scope of this Competitive Revision project will enhance the pace of discovery and it will provide additional employment for post-doctoral scientists. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) has been shown to induce osteogenesis in various cell types, including rat muscle fibroblasts, mouse cementoblasts, mouse oral mucosal cells, and mouse embryonic stem cells. Our preliminary studies have shown that LRAP stimulates osteoblastogenesis and inhibits adipogenesis of bone marrow mesenchymal stem cells (BMMSC). Canonical Wnt/2-catenin signaling pathway is activated upon LRAP treatment;while a specific Wnt inhibitor sFRP-1, completely blocks LRAP-mediated lineage selection of BMMSCs. We hypothesize that LRAP is capable of modulating the reciprocal relationship between osteogenic and adipogenic cell lineages in BMMSCs through activating the canonical Wnt/2-catenin pathway. We propose the following specific aims to test this hypothesis. Aim I: To determine whether activation of the canonical Wnt/ 2 -catenin pathway by LRAP is necessary and sufficient to stimulate osteoblastogenesis and to inhibit adipogesis of BMMSCs in vitro. Aim II: To investigate the mechanism by which LRAP activates the canonical Wnt/2-catenin pathway and to characterize the functional domain(s) in LRAP. Aim III: To examine the in vivo effect of LRAP on bone and adipose tissue in transgenic mice expressing the LRAP transgene in bone marrow and in amelogenin null mice. PUBLIC HEALTH RELEVANCE: A previously unrecognized function of amelogenin proteins is to stimulate the differentiation of stem cells towards bone formation while suppressing their differentiation into fat forming cells. This switch is activated by amelogenin stimulating the Wnt signaling cascade. Utilizing amelogenin proteins may improve surgical correction of birth defects, trauma and tumor induced bone loss by providing a means to stimulate bone formation replacing foreign materials with real bone.